Specific location of penicillin-binding proteins within the cell envelope of Escherichia coli.

This communication deals with the location of penicillin-binding proteins in the cell envelope of Escherichia coli. For this purpose, bacterial cells have been broken by various procedures and their envelopes have been fractioned. To do so, inner (cytoplasmic) and outer membranes were separated by isopycnic centrifugation in sucrose gradients. Some separation methods (Osborn et al., J. Biol. Chem. 247:3962-3972, 1972; J. Smit, Y. Kamio, and H. Nikaido, J. Bacteriol. 124:942-958, 1975) revealed that penicillin-binding proteins are not exclusively located in the inner membrane. They are also found in the outer membrane (A. Rodríguez-Tébar, J. A. Barbas, and D. Vásquez, J. Bacteriol. 161:243-248, 1985). Under the milder conditions for cell rupture used in this work, an intermembrane fraction, sedimenting between the inner and outer membrane, can be recovered from the gradients. This fraction has a high content of both penicillin-binding proteins and phospholipase B activity and may correspond to the intermembrane adhesion sites (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-769, 1982). We postulate that this intermembrane fraction is a labile structure that contains a high amount of all penicillin-binding proteins which are usually found in both the inner and outer membranes when the adhesion sites are destroyed by the cell breakage and fractionation procedures.     

Deacylation of penicilloylated penicillin-binding proteins from Micrococcus luteus: kinetic study of thiol-promoted release of the bound penicilloyl moiety

The stability of covalent complexes obtained by labelling penicillin-binding proteins 1-6 from Micrococcus luteus with a radioactive derivative of ampicillin has been examined in the presence of thiols. When the incubation medium contained only 1 mM 2-mercaptoethanol, the complexes were almost unaffected for at least 1 h. If 20 mM dithiothreitol was also included in the medium, the amount of bound radioactivity decreased throughout the incubation period. The breakdown of the complexes derived from penicillin-binding proteins 4 and 5 proceeded very slowly, following an apparent first-order kinetics, whereas the kinetics of deacylation of other penicillin-binding proteins exhibited a biphasic pattern with an initial fast phase followed by a slow one, each of which could be approximated by an apparent first-order reaction. This behavior is explained adequately by a two-step mechanism: the penicilloylated penicillin-binding proteins are first deacylated in a reversible exchange with the added thiol, giving rise to an intermediate thioester; once formed, this intermediate is hydrolysed irreversibly. A simple graphical method has been devised to deduce rate constants from the time course of the reaction. Theoretical curves have been constructed, and they fit experimental data satisfactorily. The results point out that added thiols may effectively interfere with the quantitation of penicillin-binding proteins; therefore, the stability of penicilloylated penicillin-binding proteins should be checked carefully when these protecting agents are included in membrane extracts or incubation media.     

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome

A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.     

Membrane topology of penicillin-binding protein 3 of Escherichia coli

The beta-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM beta-lactamase to random positions within the PBP3 gene were determined. Fusions of beta-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained greater than or equal to 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.     

Location of some proteins involved in peptidoglycan synthesis and cell division in the inner and outer membranes of Escherichia coli.

Inner and outer membranes of Escherichia coli were separated by isopycnic centrifugation in sucrose gradients and analyzed for the presence of penicillin-binding proteins. All penicillin-binding proteins--except penicillin-binding protein 3, which is found almost exclusively in the cytoplasmic membrane and is involved in septum formation--are also found in gradient fractions corresponding to the outer membrane. Our results support the hypothesis that approximately half of the total amount of penicillin-binding proteins may be sacculus-located proteins linked to the outer membrane, probably through peptidoglycan bridges.     

Classification of phospholipases A2 according to sequence. Evolutionary and pharmacological implications

The inhibition of elongation of Bacillus megaterium KM growing in the presence of low concentrations of nocardicin A resulted in the production of osmotically stable, actively dividing coccal-shaped cells. Saturation of penicillin-binding proteins 3a and 3b with nocardicin A in vivo at these concentrations was correlated with the inhibition of cell elongation. Analysis of the DD-carboxypeptidase activity of isolated vegetative membranes of B. megaterium KM in vitro indicated that penicillin-binding protein 4 is not a DD-carboxypeptidase under the assay conditions used. Penicillin-binding proteins were analysed by two-dimensional gel electrophoresis and the suitability of lysozyme treatment of cells as a method of membrane preparation was investigated with regard to the detection of proteins with highly labile penicillin-binding activities in vitro.     

Cephalosporin-sensitive penicillin-binding proteins of Staphylococcus aureus and Bacillus subtilis active in the conversion of [14C]penicillin G to [14C]phenylacetylglycine.

Breakdown of the covalent complex formed between [14C]penicillin G and higher molecular weight, cephalosporin-sensitive penicillin-binding proteins was studied using mixtures of the purified proteins isolated from membranes of Staphylococcus aureus and Bacillus subtilis. These penicillin-binding proteins were found to release the bound 14C label in a first order process characterized by half-lives of 10 to 300 min at 37 degrees C. Denaturation of the penicilloyl.penicillin-binding proctein complex prevented this release, indicating that the process is enzyme-catalyzed. [14C]Phenylacetylglycine was identified as the major labeled fragmentation product, indicating that these cephalosporin-sensitive penicillin-binding proteins, for which no in vitro transpeptidase or carboxypeptidase activity has been found, catalyze the same fragmentation of the bound penicilloyl moiety previously described for several penicillin-sensitive D-alanine carboxypeptidases.     

The second peptidoglycan hydrolase of Streptococcus faecium ATCC 9790 covalently binds penicillin.

A second peptidoglycan hydrolase (muramidase-2) of Streptococcus faecium ATCC 9790 (Enterococcus hirae) has been purified to apparent homogeneity. The enzyme has been shown to be a beta-1,4-N-acetylmuramoylhydrolase (muramidase; EC 3.2.1.17) and to differ in substrate specificity from a previously isolated muramidase. Purified enzyme appears as two protein staining bands with molecular masses of 125 and 75 kilodaltons (kDa) on polyacrylamide gels after sodium dodecyl sulfate electrophoresis. Elution and renaturation of protein bands from sodium dodecyl sulfate-polyacrylamide gels showed that both proteins have muramidase-2 activity. Both proteins have been shown to bind radioactive benzylpenicillin and have the same electrophoretic mobilities as penicillin-binding proteins 1 and 5 present in membrane preparations of this organism, respectively. Incubation of a [14C]penicillin G-labeled 125-kDa form of the enzyme with crude alkaline extracts from S. faecium (which did not contain added proteinase inhibitors) showed the endogenous conversion of the radiolabeled 125-kDa form to the radiolabeled 75-kDa form of the enzyme.     

Penicillin-binding proteins in Proteus species.

Penicillin-binding proteins in three species of Proteus, Proteus mirabilis, P. morganii, and P. rettgeri, were investigated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Penicillin-binding proteins in these Proteus species were compared with those in Escherichia coli K-12. An approximate correlation between penicillin-binding proteins in E. coli and those in Proteus species was shown by several criteria: electrophoretic mobilities; affinities of several beta-lactam antibiotics which show characteristic patterns of binding to penicillin-binding proteins in E. coli; relation between affinities of antibiotics to the proteins and effects on morphological changes in Proteus species; location of beta-lactamase activity among penicillin-binding proteins; and thermostability. The electrophoretic mobilities and several other characteristics of penicillin-binding proteins among the Proteus species examined were found to be similar from species to species and differed only slightly from those of E. coli.     

The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family.

Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-directed-mutagenesis experiments.     

The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding protein 1A and 1B of Escherichia coli K12

Penicillin-binding proteins 1A and 1B of Escherichia coli are the major peptidoglycan transglycosylase-transpeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764-base-pair fragment of DNA that contained the ponA gene, encoding penicillin-binding protein 1A, was determined. The sequence predicted that penicillin-binding protein 1A had a relative molecular mass of 93 500 (850 amino acids). The amino-terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758-base-pair fragment of DNA that contained the ponB gene, encoding penicillin-binding protein 1B, was also determined. Penicillin-binding protein 1B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin-binding protein 1B were predicted to have relative molecular masses of 94 100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin-binding proteins 1A and 1B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identity. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin-binding proteins 3 or 5, or to the class A or class C beta-lactamases. Two short regions of amino acid similarity were, however, found between penicillin-binding proteins 1A and 1B and the other penicillin-binding proteins and beta-lactamases. One of these included the predicted active-site serine residue which was located towards the middle of the sequences of penicillin-binding proteins 1A, 1B and 3, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The other region was 19-40 residues to the amino-terminal side of the active-site serine and may be part of a conserved penicillin-binding site in these proteins.     

Reactivity of penicillin-binding proteins with peptidoglycan-mimetic beta-lactams: what's wrong with these enzymes?

Beta-lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of their cell membrane to catalyze the final step in cell wall (peptidoglycan) synthesis. They have been classified into two major groups, one of high molecular weight, the other of low. Members of the former group act as transpeptidases in vivo, and their inhibition by beta-lactams leads to cessation of bacterial growth. The latter group consists of DD-carboxypeptidases, and their inhibition by beta-lactams is generally not fatal to bacteria. We have previously shown that representatives of the former group are ineffective at catalyzing the hydrolysis/aminolysis of peptidoglycan-mimetic peptides in vitro [Anderson et al. (2003) Biochem. J. 373, 949-955]. The theme of these experiments is expanded in the present paper where we describe the synthesis of a series of beta-lactams (penicillins and cephalosporins) containing peptidoglycan-mimetic side chains and the kinetics of their inhibition of a panel of penicillin-binding proteins spanning the major classes (Escherichia coli PBP 2 and PBP 5, Streptococcus pneumoniae PBP 1b, PBP 2x and PBP 3, the Actinomadura R39 DD-peptidase, and the Streptomyces R61 DD-peptidase). The results of these experiments mirror and expand the previous results with peptides. Neither peptides nor beta-lactams with appropriate peptidoglycan-mimetic side chains react with the solubilized constructs of membrane-bound penicillin binding proteins (the first five enzymes above) at rates exceeding those of generic analogues. Such peptides and beta-lactams do react at greatly enhanced rates with certain soluble low molecular weight enzymes (R61 and R39 DD-peptidases). The former result is unexpected and interesting. Why do the majority of penicillin-binding proteins not recognize elements of local peptidoglycan structure? Possible answers are discussed. That this question needs to be asked casts fascinating shadows on current studies of penicillin-binding proteins for new drug design.     

Staphylococcus aureus and Micrococcus luteus peptidoglycan transglycosylases that are not penicillin-binding proteins.

Major peptidoglycan transglycosylase activities, which synthesize uncross-linked peptidoglycan from lipid-linked precursors, were solubilized from the membranes of Staphylococcus aureus and Micrococcus luteus and were partially purified. The transglycosylase activities were separated from penicillin-binding proteins by solubilization and by purification steps. Therefore, we concluded that these activities were not activities of the penicillin-binding proteins, which are the presumptive peptidoglycan transpeptidases in these gram-positive cocci. Unlike Escherichia coli, in which the network structure of peptidoglycan is synthesized by multiple two-headed penicillin-binding proteins with both transpeptidase and transglycosylase activities, these gram-positive cocci have cell wall peptidoglycan which seems to be synthesized by penicillin-binding protein transpeptidases and a separate transglycosylase.     

Purification and characterization of the penicillin-binding protein that is the lethal target of penicillin in Bacillus megaterium and Bacillus licheniformis. Protein exchange and complex stability.

The penicillin-binding protein that is thought to be the lethal target of penicillin in Bacillus megaterium (protein 1) has been purified to greater than 95% homogeneity. The membrane-bound penicillin-binding proteins were solubilized with a non-ionic detergent and partially separated from each other by ion-exchange chromatography on DEAE-Sepharose CL-6B. Protein 1 was subsequently purified by covalent affinity chromatography on ampicillin-affinose. Bacillus licheniformis contains an equivalent penicillin-binding protein (protein 1) that can be more readily purified to virtual homogeneity in a one-step procedure. It was separated from the other penicillin-binding proteins by utilizing the observation that in this organism, this particular protein is the only one whose covalent complex with benzylpenicillin subsequently breaks down. Membranes were treated with saturating concentrations of benzylpenicillin followed by the removal of free penicillin and further incubation to allow the complex between benzylpenicillin and protein 1 to break down. The penicillin-binding proteins were then solubilized and applied to a column of ampicillin-affinose to which only protein 1 was bound as the other penicillin-binding proteins still had benzylpenicillin bound to them. Pure protein 1 was eluted from the affinity resin with hydroxylamine. The interaction of benzylpenicillin with purified protein 1 has been studied by separating unbound antibiotic from the benzylpenicillin . protein complex by paper electrophoresis. Benzylpenicillin reacts with the protein rapidly to form a covalent complex and the fully saturated complex has a molar ratio of bound [14C] benzylpenicillin: protein of 0.7:1. The complex breaks down, obeying first-order kinetics, with a half-life of 16 min at 35 degrees C, a value identical to that obtained with the membrane-bound protein. The concentration of benzylpenicillin that results in the formation of 50% of the maximum amount of benzylpenicillin . protein complex is that at which the molar amount of benzylpenicillin present is equal to 50% of the molar amount of penicillin-binding protein, rather than being a measure of any of the kinetic parameters of the binding reaction. This observation may be significant in the interpretation of previous results where the amounts of penicillins needed to kill cells or to inhibit penicillin-sensitive reactions have been expressed as concentrations. The possible importance of the breakdown of beta-lactam . protein complexes in the clinical use of these antibiotics is discussed.     

Biochemical and genetical approaches to the mechanism of action of penicillin

Since the discovery in 1965 that penicillin inhibits the transpeptidation reaction in peptidoglycan synthesis, a considerable effort has been put into the purification of enzymes that catalyse this reaction. This has resulted in the recognition that bacteria possess multiple forms of these penicillin-sensitive enzymes and has made it difficult to identify the precise target that penicillin inactivates to kill the organism. Recently penicillin-sensitive enzymes have been detected and studies as penicillin-binding proteins on sodium dodecyl sulphate polyacrylamide gels. The availability of this convenient method for identifying penicillin-sensitive enzymes has allowed biochemical and genetical approaches to be used to dissect their roles in the lethal effects of penicillin and other beta-lactam antibiotics. Three penicillin-binding proteins (1 B, 2 and 3) have been identified as killing targets for penicillin in Escherichia coli, whereas four other binding proteins are not implicated in the mechanism of action of the antibiotic. The complex biological effects that beta-lactam antibiotics produce on the growth of E. coli can be explained by their interaction with the three killing targets. Progress in the correlation of penicillin-binding proteins with penicillin-sensitive enzymes and in the development of strains of E. coli that overproduce penicillin-binding proteins is discussed.     

Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in Staphylococcus aureus

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly. The absolute numbers of each PBP were determined by direct measurement and have been reported for two Staphylococcus aureus strains, RN4220 (methicillin-sensitive S. aureus) and RN450M (methicillin-resistant S. aureus). From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made. These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in S. aureus.     

Fractionation of the Providencia stuartii cell envelope

Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.     

Use of a beta-lactamase fusion vector to investigate the organization of penicillin-binding protein 1B in the cytoplasmic membrane of Escherichia coli

The coding region for the mature form of TEM beta-lactamase was fused to random positions within the coding region of the penicillin-binding protein 1B (PBP 1B) gene and the nucleotide sequences across the fusion junctions of 100 in-frame fusions were determined. All fusion proteins that contained at least the NH2-terminal 94 residues of PBP 1B provided individual cells of E. coli with substantial levels of ampicillin resistance, suggesting that the beta-lactamase moiety had been translocated to the periplasm. Fusion proteins that contained less than or equal to 63 residues of PBP 1B possessed beta-lactamase activity, but could not protect single cells of E. coli from ampicillin, indicating that the beta-lactamase moiety of these fusion proteins remained in the cytoplasm. The beta-lactamase fusion approach suggested a model for the organization of PBP 1B in which the protein is embedded in the cytoplasmic membrane by a single hydrophobic transmembrane segment (residues 64-87), with a short NH2-terminal domain (residues 1-63), and the remainder of the polypeptide (residues 88-844) exposed on the periplasmic side of the cytoplasmic membrane. The proposed model for the organization of PBP 1B was supported by experiments which showed that the protein was completely digested by proteinase K added from the periplasmic side of the cytoplasmic membrane but was only slightly reduced in size by protease attack from the cytoplasmic side of the membrane.     

Active-site and membrane topology of the DD-peptidase/penicillin-binding protein no. 6 of Enterococcus hirae (Streptococcus faecium) A.T.C.C. 9790.

The membrane-bound 43,000-Mr penicillin-binding protein no. 6 (PBP6) of Enterococcus hirae consists of a 30,000-Mr DD-peptidase/penicillin-binding domain and a approximately 130-residue C-terminal appendage. Removal of this appendage by trypsin proteolysis has no marked effect on the catalytic activity and penicillin-binding capacity of the PBP. Anchorage of the PBP in the membrane appears to be mediated by a short 15-20-residue stretch at the C-terminal end of the appendage. The sequence of the 50-residue N-terminal region of the PBP shows high degree of homology with the sequences of the corresponding regions of the PBPs5 of Escherichia coli and Bacillus subtilis. On this basis the active-site serine residue occurs at position 35 in the enterococcal PBP.     

Activities and regulation of peptidoglycan synthases

Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.